This invention relates to a method for regulating expression of at least one virulence gene of Agrobacteria. In particular, this invention relates to the stimulation of embryonic cereal cells for the production of phenolic and/or other compounds. In addition, this invention relates to the use of the secreted phenolic and/or other compounds for activation of the vir-operon of Agrobacteria resulting in transformation of the embryonic cereal cells.
Agrobacterium tumefaciens has been extensively exploited as an important gene delivery tool for most of the families of higher plants. Under laboratory conditions, it has been shown that the host range of Agrobacterium can be extended to include virtually any living cell, for example, other prokaryotes like Streptomyces lividans, yeast, fungi and cultured human cells.
Agrobacterium achieves the transformation of its hosts by transferring a well defined segment of DNA (called transfer DNA (T-DNA)) from its tumour inducing (Ti) plasmid to host cells. The transfer process requires a number of components: chromosomal and Ti plasmid-encoded gene products. Virulence (vir) genes are contained within the Ti (tumour inducing) plasmid and encode proteins required for processing and transfer of T-DNA.
With respect to plant transformation, the vir region of the Ti-plasmid is activated by a two component system, Vir A/Vir G and a galactose binding protein ChvE in response to phenolic compounds and sugars exuded from wounded plant cells. In a manner similar to bacterial two-component systems, Vir A, a periplasmic membrane-spanning protein, senses the phenolic compound(s)/simple monosaccharide stimuli and autophosphorylates at its histidine residue in the cytoplasmic C-terminus. The Vir G protein is considered to act as a signal-response regulator, because after receiving a phosphate (through transphosphorylation) from the Vir A phospho-histidine (Vir A sensor kinase), it activates transcription of the vir genes by binding to the vir boxes in the promoters of vir genes and activates their gene expression. This enables the processing and transport of the T-DNA through a T-pilus-associated type IV secretion system (T4SS) from Agrobacterium into the plant nuclear genome where the T-DNA is integrated into chromosomal DNA, thus completing the transfer of any transgenes that might have been cloned within the T-DNA boarders of Agrobacterium tumefaciens. 
Acetosyringone, a plant cell wound product, is one of the major plant phenolic inducers of the two-component Vir A/Vir G system in Agrobacterium tumefaciens. Other secreted plant diffusible factors which induce T-DNA circularization and vir gene expression have been identified and include small (<100 Da) diffusible plant metabolites produced by actively metabolizing plant cells, catecol, sugars and amino acids. Many of these factors are produced both in monocotyledonous and dicotyledonous plants, and this partially explains why some monocotyledonous plants are susceptible to Agrobacterium tumefaciens. There is, however, a distinction between the ability of these factors to act as chemo-attractants to Agrobacterium (a phenomenon that brings the bacterium within close proximity to susceptible host cells) and the ability to induce vir gene expression, which is required for T-DNA transfer. Chemo-attraction is very sensitive and occurs at low molar concentrations of the diffusible products, whereas higher concentrations (as occurs in the proximity of secreting cells) of the products are required to induce vir gene expression. This perhaps explains why many monocotyledonous plants have been regarded as recalcitrant to Agrobacterium tumefaciens-mediated transformation, and often require supplementation with synthetic acetosyringone when Agrobacterium is employed as a preferred vehicle for transformation. In rice, for example, supplementation with acetosyringone is required and contributes to higher T-DNA transfer if used at the early initial stages of bacterial infection and also during co-cultivation. For wheat, higher amounts of acetosyringone (200 μM instead of the usual 100 μM) lead to higher levels of T-DNA transfer and transformation efficiency. Studies with barley revealed that the effects of acetosyringone on transformation efficiency are dependent on the plant species concerned. In the case of barley, acetosyringone concentrations in the range between 200-1000 mg/l (higher than for rice) can be used effectively. There are a few exceptions within monocotyledonous plants, depending on the target explant tissue used for transformation, where the addition of acetosyringone may not be effective. In lillies (ornamental monocotyledonous plants), for example, the addition of acetosyringone does not have any effect on transformation efficiency. Generally, it is well documented that wounded dicotyledonous plant tissues produce phenolic compounds such as acetosyringone (4-acetyl-2,6-dimethoxyphenol), and that monocotyledonous plants may fall into two categories: those that do not produce phenolic compounds and those which may produce acetosyringone despite the levels being so low as not to affect vir gene induction.
In certain instances, acetosyringone alone may not efficiently induce vir genes in Agrobacterium tumefaciens. It has been shown that when the concentration of acetosyringone is limited (as occurs in some monocotyledonous plants), a group of aldoses (for example: L-arabinose, D-xylose, D-glucose, D-mannose, D-idose, D-galactose and D-talose) can effectively enhance acetosyringone-dependent expression of vir genes. This suggests that other factors, sugars in this particular case, directly enhance a signaling process initiated by phenolic inducers to result in an increase in the expression of vir genes of Agrobacterium. The protein ChvE, which binds glucose-galactose and interacts with the vir A protein, was specifically identified and implicated in broadening the phenolic recognition profiles of Agrobacterium vir A protein and hence vir gene expression, especially when it was available at high levels. Other enhancers of vir gene expression include phosphate starvation and acidic culture medium. When acetosyringone alone is used to induce vir gene expression, Agrobacterium transformation efficiency for sorghum is very low, even when the current best protocols are used (Zhao et al., 2000. Plant Mol. Biol. 44: 789-798).
U.S. Pat. No. 5,641,644 teaches a method for reproducibly and efficiently transforming the genome of a monocotyledonous plant, and in particular a gramineous plant such as a cereal. This disclosure concentrates on the use of compact embryogenic callus or intact tissue capable of forming compact embryogenic callus. Transformation by this method is, however, limited to electroporation and is silent regarding Agrobacterium-mediated transformation. Furthermore, no mention is made regarding the use of endogenous compounds to facilitate and enhance transformation.
The methodology taught in U.S. Pat. No. 5,641,644 was subsequently extended to cereal crops in general (U.S. Pat. No. 5,712,135), and specifically to rice (U.S. Pat. No. 6,002,070).
The applicants have therefore identified a need to increase the Agrobacterium-mediated transformation efficiency of cereals, especially sorghum.